Molecular insights into pattern recognition and apoptosis in fish under pathological conditions through characterization of Toll-like receptor 2 and caspase 3 from Rock bream

Abstract

Pattern recognition and apoptosis can be considered as significant bio-machineries in physiology of multicellular organisms which essentially contribute to their survival and development, even in the grievous environments. According to the recent reports, these two processes were found to be interrelated, especially in pathogenic infections. Upon the recognition of molecular patterns of invading pathogens and damaged cells, pattern recognition receptors (PRRs) can mount immune responses, via activating different downstream signaling molecules to initiate various immune signaling cascades, which can trigger the death cascade pathways to stimulate apoptosis as a host defensive mechanism to reduce the pool of infected cells, in order to restrict the propagation of the infection. With respect to the pattern recognition receptors, Toll-like receptors (TLRs) are known to be the initially discovered and extensively studied group of PRRs, which can interact with wide array of ligands in invaded pathogens. In terms of apoptosis, caspases as cysteine aspartic acid proteases can be considered as key molecular mediators to propagate the death signal through a cascade. Among them caspase 3 plays a pivotal role as a molecular hub in exclusionary phase of apoptosis by linking different death signaling pathways together to direct cells toward death. In this study a novel counter parts of TLR 2 (RbTLR2) and Caspase 3 (Rbcasp3) genes, from an economically important teleost species, rock bream (Oplegnathus fasciatus), was characterized at genomic level, while analyzing their transcriptional modulation under physiological and pathological conditions. The complete genomic sequences of both RbTLR2 and Rbcasp3 genes were identified using a random sheared bacterial artificial chromosome (BAC) based gDNA library. Subsequently, the identified sequences were characterized using bioinformatics tools, while comparing the sequences with other known similitudes usingmultiple and pairwise sequence alignment strategies of ClustalW software. In order to determine the evolutionary position of both molecules, phylogenetic relationship was determined with their known orthologues. Moreover, genomic organization of both Rbcasp3 and RbTLR2 as exons and introns was determined and compared with the known genomic architectures of their counterparts, further anticipating the respective promoter regions with their potential transcriptional factor binding sites. In addition, tertiary structure of Rbcasp3 was modeled using I-TASSER online server and visualized using RasMol 2.7.5.2 software to predict the structure-function relationship. Furthermore, Rbcasp3 complete coding sequence was cloned into pMAL-c2X expression vector and subsequently expressed and purified the recombinant protein to demonstrate the proteolytic activity of recombinant Rbcasp3 (rRbcasp3). The tissue specific transcriptional profile of both genes were determined using quantitative real time PCR (qRT-PCR), further analyzing their transcriptional regulation in liver tissues, upon the inductions with pathogenic stimulants and their molecular patterns. The complete cDNA sequences of RbTLR2 (4399 bp) and Rbcasp3 (2756 bp) were consisted of 2451 bp and 849 bp coding sequences, encoding polypeptides of 817 and 283 amino acids, respectively. RbTLR2 was predicted to be comprised of a signal peptide whereas signal sequence was absent in Rbcasp3. Predicted molecular masses of RbTLR2 and Rbcasp3 were around 92 kDa and 31 kDa respectively. RbTLR2 resembled the typical TLR family signature whereas Rbcasp3 demonstrated the characteristic caspase domain architecture. TLR 2 orthologue of orange spotted grouper (Epinephelus coioides) exhibited the maximum identity and similarity values with RbTLR2. On the other hand, Rbcasp3 shared the highest sequence compatibility with its orthologue of large yellow croaker (pseudosciaena crocea). According to the multiple sequence alignment, C-terminal region of RbTLR2 including consensus sequences in TIR domain showed thorough conservation among its vertebrate counterparts and Rbcasp3 showed well conserved motifs among its orthologues, includingpenta peptide motif and RGD motif. Phylogenetic analysis of RbTLR2 evidenced to its higher evolutionary proximity with orthologue from orange spotted grouper, among the other vertebrate similitudes, whereas Rbcasp3 demonstrated a closer evolutionary relationship with its caspase 3 homologue of large yellow croaker lying with in the fish clade. The modeled three dimensional structure of Rbcasp3 resembled the characteristic features of the folding of human caspase 3 tertiary structure, validating its in-silico derived primary structure. Anticipated promoter regions of both RbTLR2 and Rbcasp3 genes consisting different transcriptional factor binding sites, most of which are related with immune signaling, revealed the tight regulation of their transcription by external and internal environmental factors. Moreover, genomic length of RbTLR2 was 10849 bp and genomic architecture was composed of multi-exonic structure, containing 11 exons interrupted by 10 introns. In contrast, Rbcasp3 was consisted of 6 exons, interrupted by 5 introns, expanded in 7529 bp genomic length. Arrangements at genomic level in both genes shared substantial compatibility with known genomic architectures of respective fish counterparts. Rbcasp3 gene was cloned into pMAL-c2X, transformed into BL21 (DE3) cells and induced with IPTG to overexpress the proteins. The recombinant proteins were purified in MBP-fused form using an affinity chromatography and used in protease activity assay against caspase 3/7 (DEVD-pNA), caspase 8 (IETD-pNA) and caspase 9 (LEHD-pNA) specific substrates. Recombinant protein demonstrated a detectable and specific cysteine-aspartate protease activity against DEVD-pNA, convincing the functional viability of Rbcasp3. RbTLR2 and Rbcasp3 demonstrated a different but ubiquitous transcriptional profile in tissues examined in healthy fish, where higher levels of mRNA were detected in spleen and liver, respectively. According to the immune challenge experiments in liver tissues of rock bream, transcription of both genes got up-regulated upon different stimuli, with the time.Streptococcus iniae induction could boost RbTLR2 transcript level more prominently. Moreover, Edwardsiella tarda and LPS also significantly (p < 0.05) induced the transcription of RbTLR2 in liver tissues. On the other hand, two viral stimuli, polyinosinic-polycytidylic acid (poly I:C) and rock bream iridovirus (RBIV) could trigger the augmentation of RbTLR2, where the up-regulations upon RBIV convinces the recognition of PAMPs of DNA virus at the first time in teleosts. Stimulation with lipopolysaccharides (LPS) caused prolonged up-regulation of Rbcasp3 mRNA level, whereas Edwardsiella tarda stimulated its late-phase significant (P < 0.05) transcriptional induction. Rock bream iridovirus (RBIV) up-regulated Rbcasp3 transcription significantly (P < 0.05) at late-phase, however poly I:C induced the transcription of Rbcasp3 significantly at early-phase of the experiment. Collectively, this study perceives molecular perspectives of apoptosis and pattern recognition of fish, considering rock bream as a model organism, while characterizing the crucial elements of respective processes, TLR2 and caspase 3. Moreover, this furnished a substantial contribution to the extension of existing knowledge on innate immune mechanisms, with respect to the pathogen recognition and apoptosis, emphasizing the requirement of further investigations and mining of different components of these processes in teleost species.